Nuclear transfer methods have been developed and used successfully to produce cloned sheep, cattle, mice, goats and pigs. Two cell components are combined to produce a cloned embryo; the donor nuclear genome (karyoplast) that is the target for clonal replication, and the enucleated oocyte (cytoplast) whose cytoplasmic constituency is sufficiently competent to facilitate genome reprogramming and support embryonic development to term.
Mammalian oocyte cytoplasts have been prepared by physically removing nuclear chromatin by micromanipulation techniques in preparation to receive the donor genome. Enucleated oocytes arrested at metaphase of meiosis II (MII) are subsequently “reconstructed” by the addition of the donor karyoplast typically using either electrofusion or microinjection techniques. However, physical enucleation is generally technically demanding, time consuming, inherently invasive and clearly damaging to cytoplast spatial organization. Moreover, in certain instances, development of reconstructed embryos is inherently inefficient.
One alternative strategy to physical enucleation has been to treat oocytes with agents that modify the processes of karyokinesis and cytokinesis and result in chemically enucleated oocytes at high rates (>85%). However, certain studies have reported that exposure of metaphase I and MII oocytes to etoposide, a topoisomerase II inhibitor, and cycloheximide yields enucleated cytoplasts with limited ability to support cleavage or blastocyst development, and term development of reconstructed embryos has not been reported.
Hence, a need exists for improved methods for developing nuclear transfer embryos.